PROJECT SUMMARY We recently found that mutations in ETV6 lead to thrombocytopenia with bleeding diathesis, red cell macrocytosis, and predisposition to leukemia. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.P214L mutation and non-affected family members. Additionally, single cell RNA-sequence of peripheral mononuclear blood cells from these patients demonstrated significant changes in the expression patterns of mRNAs of Interferon (IFN) Response Genes, suggesting a critical role for ETV6 in maintaining bone marrow homeostasis. This proposal will test the central hypothesis that normal regulation and function of ETV6 is essential for transcriptional events that control MK differentiation and formation of platelets that function properly under homeostatic and inflammatory conditions. We generated a transgenic mouse in which Etv6 exhibits the p.P214L mutation at the mouse orthologue conserved position (Etv6P214L). Mice with this mutation (Etv6P214L) have reduced platelet counts and exhibit a platelet defect. In this proposal, we will test three aims that will determine the mechanisms by which ETV6 regulates critical functions of MKs and platelets. In Specific Aim 1 we will define roles for ETV6 in regulating MK differentiation, platelet formation and platelet function. For this purpose, we will use Etv6P214L, Etv6-/- Gata1-Cre and Etv6-/- Pf4-Cre mice to determine the effects of Etv6 gene disruption in MK progenitors and MKs. These results will be compared to MK differentiation and proplatelet formation in CD34+- derived MK that are cultured from patients with ETV6 mutations. Additionally, we will study in vitro and in vivo platelet responses from these mice. In Specific Aim 2 we will delineate the contributions of ETV6 in modulating transcriptional events in MK. This aim will test the hypothesis that ETV6 directly regulates transcriptional events in MKs. We will identify Etv6-dependent gene candidates by performing RNA-seq in MKs and platelets isolated from Etv6P214L, Etv6-/-Gata1-Cre, and Etv6-/- Pf4-Cre mice. These results will be compared to Chip-seq/DNase I seq data and results obtained from mice and human MKs that carry the ETV6 p.P214L and R418G mutations. We will determine the mechanisms by which ETV6 regulates the transcription of candidate mRNAs by identifying ETV6 effectors and test them functionally. Finally, in Specific Aim 3 we will determine the consequences of ETV6 disruption on IFN response genes in bone marrow homeostasis. This aim will test the hypothesis that ETV6 regulates IFN response genes by interacting with HDAC3, and disruption of ETV6 function will generate a proinflammatory milieu that affects normal megakaryopoiesis and hematopoiesis in general. Discoveries from this application will further advance our understanding of MK and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function.